Dependence on p38 MAPK signalling in the up-regulation of TLR2, TLR4 and TLR9 gene expression in Trichomonas vaginalis-treated HeLa cells

Abstract

Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that recognize conserved pathogen-associated molecular patterns (PAMPs) synthesized by micro-organisms. Despite the essential requirement for TLRs in prokaryotic infection, the pattern and regulation of TLR gene expression by Trichomonas vaginalis in the mucocutaneous barrier are still unknown. Our hypothesis is that T. vaginalis-infected epithelial cells are major effector cells in the skin barrier. These cells function as a central regulator of TLR gene expression, thus accelerating the process of barrier dysfunction via increased release of chemokines and proinflammatory cytokines. To test this hypothesis, RT-PCR was performed on TLRs, interleukin (IL)-8 and tumour necrosis factor (TNF)-alpha. Stimulation of HeLa cells by T. vaginalis was observed to up-regulate TLR2, 4 and 9 mRNA expression as well as that of IL-8 and TNF-alpha. To further clarify the molecular mechanism of barrier devastation triggered by these up-regulatory stimuli, we examined the profiles of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappaB activation in HeLa cells using specific inhibitors. Interestingly, pretreatment of HeLa cells with the p38 MAPK inhibitor SB203580 demonstrated inhibition of T. vaginalis-induced up-regulation of TLR2, 4, and 9 mRNA expression. By contrast, inhibition of ERK or NF-kappaB activation failed to block T. vaginalis-induced up-regulation of TLR9 mRNA expression or TLR2 and TLR4 mRNA expression, respectively. In addition, pretreatment with SB203580 reduced epithelium-derived IL-8 and TNF-alpha release evoked by T. vaginalis. Our results show that T. vaginalis infection of the mucocutaneous barrier could up-regulate TLR2, 4 and 9 gene expression via the p38 MAPK signalling pathway in epithelial cells; this process then leads to modulation of p38 MAPK-dependent IL-8 and TNF-alpha release from the epithelium.

Figure 1

Up-regulation of Toll-like receptor 2 (TLR2), TLR4 and TLR9 mRNA expression by Trichomonas vaginalis in HeLa cells. HeLa cells were treated with T. vaginalis for 1, 4, 7, 10 and 13 hr. (−) stands for T. vaginalis non-treated cells. After T. vaginalis treatment, total RNA was prepared and TLR gene expression was examined by RT-PCR. GAPDH expression was used as a control. Similar results were obtained in three independent experiments.

Figure 2

The time–course of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB activation in response to Trichomonas vaginalis infection. (a) Cells were treated with T. vaginalis for 1–13 hr. Lysates were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with anti-phospho-p38 (anti-p-p38), anti-phospho-ERK1/2, and anti-p65 antibodies. (b) T. vaginalis-induced activation of ERK, p38 MAPK and NF-κB was inhibited by 50 µm PD98059, 10 µm SB203580 or 15 µm pyrrolidinecarbodithioic (PDTC). HeLa cells were pretreated with or without PD98059, SB203580 or PDTC for 1 hr, and then treated with T. vaginalis for the indicated times. Lysates were subjected to SDS-PAGE and immunoblotted with anti-phospho-p38, anti-phospho-ERK1/2 and anti-p65 antibodies. Equal amounts of protein were loaded in each lane, confirmed by immunoblotting with anti-β-actin antibody. N, T. vaginalis and inhibitors non-treated cells.

Figure 3

The involvement of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB pathways in the regulation of TLR2, 4 and 9 mRNA expression by Trichomonas vaginalis in HeLa cells. HeLa cells were pretreated with or without 50 µm PD98059 (PD), 10 µm SB203580 (SB) or 15 µm pyrrolidinecarbodithioic (PT) for 1 hr, and then treated with T. vaginalis for 10 hr. After T. vaginalis treatment, total RNA was prepared and Toll-like receptor (TLR) gene expression was examined by RT-PCR. GAPDH expression was used as a control. Similar results were obtained in three independent experiments. −, T. vaginalis non-treated cells; N, inhibitors non-treated cells.

Figure 4

The p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 attenuates Trichomonas vaginalis-induced cytokine production. (a) HeLa cells were treated for increasing times with lipopolysaccharide (LPS) or T. vaginalis. After T. vaginalis treatment, total RNA was prepared and interleukin-8 (IL-8) and tumour necrosis factor-α (TNF-α) mRNA expression was examined by RT-PCR. GAPDH expression was used as a control. Similar results were obtained in three independent experiments. (b) HeLa cells were pretreated with or without 50 µm PD98059 (PD), 10 µm SB203580 (SB) or 15 µm pyrrolidinecarbodithioic (PDTC) for 1 hr, and then treated with T. vaginalis (TV) for 10 hr. Culture supernatants were analysed by enzyme-linked immunosorbent assay (ELISA) for IL-8 or TNF-α protein expression (pg/ml). ND stands for not detected. The average of three separate cell platings (n = 3) was determined (± standard error of the mean). The experiment was repeated three times with similar results. Asterisks indicate statistically significant decreases in IL-8 and TNF-α production induced by treatment with 15 µm SB203580 (P < 0·05). (c) HeLa cells were pretreated with or without 50 µm PD98059 (PD), 10 µm SB203580 (SB) or 15 µm PDTC (PT) for 1 hr, and then treated with T. vaginalis for 10 hr. After LPS or T. vaginalis treatment, total RNA was prepared and IL-8 and TNF-α mRNA expression was examined by RT-PCR. GAPDH expression was used as a control. Similar results were obtained in three independent experiments. N, LPS and T. vaginalis non-treated cells.